Monoclonal antibodies against antithrombin beta

ABSTRACT

This patent document relates to antibodies, antigen-binding antibody fragments (Fabs), and other protein scaffolds, directed against human antithrombin β complexed with heparin and/or heparin-like structure (ATβH). These ATβH binding proteins can block the anti-coagulant activity of ATβ to induce coagulation. Therapeutic uses of these antibodies and binders are described herein as are methods of panning and screening specific antibodies.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is being filed on 14 Mar. 2014, as a PCT Internationalpatent application, and claims priority to U.S. Provisional PatentApplication No. 61/784,590, filed Mar. 14, 2013, the entire disclosureof which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING SUBMISSION

The present application includes a Sequence Listing in electronic formatas a txt file titled “SEQUENCE-LISTING-17207.0006WOU2” which was createdon Mar. 14, 2014 and which has a size of 65.1 kilobytes (KB). Thecontents of txt file “SEQUENCE-LISTING-17207.0006WOU2” are incorporatedby reference herein.

BACKGROUND

Current unmet medical needs in the hemophilia field are mainly: (1)treatment of hemophilia patients with inhibitors (˜30% of hemophiliapatients); and (2) long acting and efficacious coagulant factors(FVIII/FIX) and/or their replacement (bypass drugs) (WFH report 2012,Paris). The most widely used bypass drug for treating hemophiliapatients with inhibitors is rFVII, which has major drawbacks such asrisk of thrombogenicity, short half-life in plasma and high productioncost. Antibodies against anti-coagulant factors, such as Tissue FactorProtein Inhibitor (TFPI), APC (Activated Protein C) and Antithrombin(AT) represent a new treatment paradigm. These antibodies not onlybypass or reduce the need for FVIII or FIX coagulation factors inhemophilia patients with inhibitors, but also exhibit longer plasmahalf-life (which reduces the dosing frequency) and, thus, increasespatient compliance. To date, there have been several antibody-basedprocoagulant drugs at the preclinical development or research stage,such as anti-TFPI and anti-APC.

AT is a major anticoagulant in human plasma. It inhibits thrombin, FXaand other serine proteases functioning in the coagulation pathway. Itconsists of 432 amino acids, is produced by the liver hepatocyte and hasa long plasma half-life of three days (Cotten, Schetz et al. 1977). Theamino acid sequence of AT is well-conserved and the homology among cow,sheep, rabbit, mouse and human is 84%-89% (Olson and Bjork 1994).Although the primary physiological targets of AT are thrombin and FXa,AT also inhibits FIXa, FXIa, FXIIa, as well as FVIIa to a lesser extent.AT exerts its inhibition together with heparin. In presence of heparinthe inhibition rate of thrombin and FXa by AT increases by 3 to 4 ordersof magnitude from 7-11×10³M⁻¹s⁻¹ to 1.5-4×10⁷ M⁻¹s⁻¹ and from 2.5×10⁻³M⁻¹S⁻¹ to 1.25-2.5 M⁻¹s⁻¹ respectively (Olson, Swanson et al. 2004).

Unlike TFPI and APC which inhibit coagulation solely at the initiatingstage and the amplification stage respectively, AT exerts its inhibitionon coagulation at both the initiation and amplification stage.Therefore, blocking AT could have more potent pro-coagulant effect thanblocking either TFPI or APC alone. Decreased AT levels and activity havebeen shown to correlate with increased thrombosis in human. Patientswith AT deficiency tend to show recurrent venous thrombosis andpulmonary embolisms (van Boven and Lane 1997). Furthermore, homozygousAT knockout mice die in the embryonic stage with an extremehypercoagulable state (Ishiguro, Kojima et al. 2000). A recent studyshows that heterozygous AT knockout hema mice in which AT is reduced by50% significantly have less blood loss and enhanced thrombin generationin a tail-clip bleeding model (Bolliger, Szlam et al. 2010).

AT is a glycoprotein with two isoforms based on differentialglycosylation on Asn135, ATα and ATβ (Bjork 1997). ATβ lacksglycosylation at Asn135 and is a minor glyco-isoform representing 10% ofhuman plasma AT. Asn135 is located adjacent to the initial heparinattachment site and constitutes part of extended heparin binding siteafter allosteric activation and D helix extension (dela. Cruz,Jairajpuri et al. 2006). The lack of bulky-sized glycan at Asn135affects ATβ activation profoundly in two ways: 1) a faster allostericactivation upon heparin binding required for inhibition of FXa and FIXa;and 2) extra accessible binding sites for higher affinity heparinbinding for inhibition of FXa and thrombin by a bridging mechanism.Indeed, under physiological salt concentration, plasma-derived ATβ bindsto heparin with a K_(D) of 36+/−3 nm while ATα binds to heparin with aK_(D) of 500+/−50 nm (Turk I V. et at., 1993). The higher affinity ofATβ for heparin leads to its preferential distribution to thesub-endothelial layer which is enriched in the heparin-likestructure—glycosaminoglycan. Consequently, ATβ is proposed to play amajor and potent role in inhibition of FXa and thrombin at the vascularinjury sites (Carlson and Atencio 1982; McCoy A J, Pei X Y, et al. 2003;Turk B, Brieditis I. et al. 1997; Witmer M R, Hatton M W. 1991;Frebelius S, et al. 1996). The importance and stronger potency of ATβrelative to that of ATα is also reported in clinical studies. Inpatients, the severity of AT homozygous mutations defective inheparin-binding is ameliorated by the beta form of AT(Martinez-Martinez, Navarro-Fernandez et al. 2012). In another study, aborderline level (˜70% of normal AT antigen and activity) of AT iscompensated by the 20%˜30% ATβ in plasma (Bayston, Tripodi et al. 1999).

SUMMARY

Monoclonal antibodies to human ATβH (ATβ complexed with heparin and/orheparin-like structure) are provided. In at least one embodiment, theanti-ATβH monoclonal antibodies exhibit binding to ATβ complexed withHeparin.

In other embodiments, the monoclonal antibodies to ATβH may beoptimized, for example to have increased affinity or increasedfunctional activity. Also provided are specific epitopes that may be onhuman ATβH and are bound by an isolated monoclonal antibody. Furtherprovided are the isolated nucleic acid molecules encoding the same.

Pharmaceutical compositions comprising the anti-ATβH monoclonalantibodies and methods of treatment of genetic and acquired deficienciesor defects in coagulation such as hemophilia A and B are also provided.

Also provided are methods for shortening bleeding time by administeringan anti-ATβH monoclonal antibody to a patient in need thereof. Methodsfor producing a monoclonal antibody that binds human ATβH are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings or claims in any way.

FIG. 1 shows a schematic representation of ATβ bound to heparin and thevarious binding domains of ATβ.

FIGS. 2A-2C show how ATβ is distinguished from ATα by lacking of oneN-glycan.

FIGS. 3A-3D show ATβ with faster binding to heparin and more potentinhibition than ATα.

FIG. 4A shows biotinlayted hAT and rAT are functional in inhibition ofFxa generation (FIG. 4A). FIGS. 4B-4C show various strategies forantibody discovery by phage display.

FIG. 5 shows a screening method for identifying antibodies capable offunctional inhibition of ATβ::heparin.

FIGS. 6A and 6B shows alignment of the amino acid sequences of the lightchain domain and heavy chain domain, respectively, of antibodiesTPP-2009 (SEQ ID NO:1 and SEQ ID NO: 2, respectively), TPP-2015 (SEQ IDNO:3 and SEQ ID NO:4, respectively), TPP-2016 (SEQ ID NO:5 and SEQ IDNO:6 respectively), TPP-2019 (SEQ ID NO:7 and SEQ ID NO:8,respectively), and TPP-2803 (SEQ ID NO:9 and SEQ ID NO:10,respectively).

FIGS. 7A-7C show antibody binding specificity determined by Biacore(FIG. 7A) and ELISA (FIG. 7B) tests, and antibody binding affinity tohuman AtβH (FIG. 7C).

FIG. 8A is a graphical representation of the effect of TPP antibodies onthrombin generation in human HEM-A plasma, and illustrates that antibodypresence increases peak thrombin generation in human HEM-A plasma.

FIG. 8B is a table showing antibodies shorten clotting time in humanHemA plasma and in human AT-deficient plasma spiked in with Atβ or Atα.

FIG. 9 is a graphical representation of the PK of antibody TPP 2009 inHEM-A mice using IV dosing at 0.3, 3 and 30 mg/kg, three mice per timepoint (10 time points over 21 days), and associated PK parameters.

FIGS. 10A and 10B show an experimental protocol for a tail veintransection (TVT) model in HemA and the efficacy of antibody TPP-2009 inthe TVT model in HemA mice. FIG. 10B shows the antibody TPP-2009 haspotent efficacy in the Tail Vein Transection (TVT) model of HemA mice.

FIGS. 11A and 11B shows a molecular model of the three-dimensionalstructures of native ATβ complexed with/without heparin (FIG. 11A), andfully activated antibody TPP2009 bound to heparin (FIG. 11B) and itspredicted epitope structure. Helix D is extended upon heparin binding B.

FIG. 12 shows a TPP2803 exhibited dose-dependent shortening of theclotting time in both normal human plasma and hemophilia patient plasmausing the FXa activated clotting assay. CT: clotting time, HEM-A:Hemophilia A plasma.

FIG. 13 shows an experimental design of the heparinized rabbit bleedingmodel; Experimental groups: Vehicle, PBS; Positive control, Protaminesulfate, (28 mg/kg IV); Negative control, M14 IgG2; treatment: 30 mg/kg;TPP2803, 3 mg/kg; TPP2803, 30 mg/kg.

FIG. 14. shows the effect of a control and TPP2803 on bleeding timebefore and after LMWH and compound administration in a heparinizedrabbit bleeding model.

FIG. 15. shows the effect of a control and TPP2803 on delta bleedingtime Significantly different from PBS (p≤0.05; T-test).

FIG. 16. shows the effect of a control and TPP2803 on blood loss beforeand after LMWH and antibody administration. (Significance by theT-test).

DETAILED DESCRIPTION

This disclosure provides antibodies, including monoclonal antibodies andother binding proteins that specifically bind to the activated form ofATβ, but exhibit comparatively little or no reactivity against the ATαform, either naïve or activated.

Definitions

For the purpose of interpreting this specification, the followingdefinitions will apply. In the event that any definition set forth belowconflicts with the usage of that word in any other document, includingany document incorporated herein by reference, the definition set forthbelow shall always control for purposes of interpreting thisspecification and its associated claims unless a contrary meaning isclearly intended (for example in the document where the term isoriginally used).

Whenever appropriate, terms used in the singular will also include theplural and vice versa. The use of “a” herein means “one or more” unlessstated otherwise or where the use of “one or more” is clearlyinappropriate. The use of “or” means “and/or” unless stated otherwise.The use of “comprise,” “comprises,” “comprising,” “include,” “includes,”and “including” are interchangeable and are not limiting. The terms“such as,” “for example,” and “e.g.” also are not intended to belimiting. For example, the term “including” shall mean “including, butnot limited to.”

As used herein, the term “about” refers to +/−10% of the unit valueprovided. As used herein, the term “substantially” refers to thequalitative condition of exhibiting a total or approximate degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, achieve or avoid an absolute result because of the manyvariables that affect testing, production, and storage of biological andchemical compositions and materials, and because of the inherent errorin the instruments and equipment used in the testing, production, andstorage of biological and chemical compositions and materials. The termsubstantially is therefore used herein to capture the potential lack ofcompleteness inherent in many biological and chemical phenomena.

The term “ATβ” or “ATβH” as used herein refers to any variant, isoform,and/or species homolog of AT in its form that is naturally expressed bycells and present in plasma and is distinct from ATα. Further, the term“ATβ” or “ATβH” as used herein can also refer to an activated form ofATβ complexed with heparin or a heparin-like structure.

The term “antibody” as used herein refers to a whole antibody and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. This term includes a full-length immunoglobulin molecule(e.g., an IgG antibody) that is naturally occurring or formed by normalimmunoglobulin gene fragment recombinatorial processes, or animmunologically active portion of an immunoglobulin molecule, such as anantibody fragment, that retains the specific binding activity.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. For example, ananti-ATβH monoclonal antibody fragment binds to an epitope of ATβH. Theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VII and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody; (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a V_(H) domain; (vi) an isolated complementarily determining region(CDR); (vii) minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., Ill et al., Protein Eng 1997;10:949-57); (viii) camelIgG; and (ix) IgNAR. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are analyzed for utility in the same manner as are intactantibodies.

Furthermore, it is contemplated that an antigen binding fragment can beencompassed in an antibody mimetic. The term “antibody mimetic” or“mimetic” as used herein refers to a protein that exhibits bindingactivity similar to a particular antibody but is a smaller alternativeantibody or a non-antibody protein. Such antibody mimetic can becomprised in a scaffold. The term “scaffold” refers to a polypeptideplatform for the engineering of new products with tailored functions andcharacteristics.

The term “anti-ATβ antibody” as used herein refers to an antibody thatspecifically binds to an epitope of ATβ associated with heparin orheparin-like. When bound in vivo to an epitope of ATβH, the anti-ATβantibodies disclosed herein augment one or more aspects of the bloodclotting cascade.

The terms “inhibits binding” and “blocks binding” (e.g., referring toinhibition/blocking of binding of ATβ substrate to ATβH) as used hereinare used interchangeably and encompass both partial and completeinhibition or blocking of a protein with its substrate, such as aninhibition or blocking by at least about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

In reference to the inhibition and/or blocking of binding of ATβsubstrate to ATβ, the terms inhibition and blocking also include anymeasurable decrease in the binding affinity of ATβ and/or ATβH to aphysiological substrate when in contact with an anti-ATβ antibody ascompared to ATβ not in contact with an anti-ATβ antibody, e.g., theblocking of the interaction of ATβ with its substrates by at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100%.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to antibody molecules of single Molecular composition.A monoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” as used herein refers to antibodies displaying asingle binding specificity that have variable and constant regionsderived from human germline immunoglobulin sequences. The humanantibodies can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).

The term “isolated antibody” as used herein is intended to refer to anantibody which is substantially free of other biological molecules,including antibodies having different antigenic specificities (e.g., anisolated antibody that binds to ATβH is substantially free of antibodiesthat bind antigens other than ATβH). In some embodiments, the isolatedantibody is at least about 75%, about 80%, about 90%, about 95%, about97%, about 99%, about 99.9% or about 100% pure by dry weight. In someembodiments, purity can be measured by a method such as columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Anisolated antibody that binds to an epitope, isoform or variant of humanATβH can, however, have cross-reactivity to other related antigens,e.g., from other species (e.g., ATβH species homologs). Moreover, anisolated antibody can be substantially free of other cellular materialand/or chemicals.

The term “specific binding” as used herein refers to antibody binding toa predetermined antigen. An antibody that exhibits specific bindingtypically binds to an antigen with an affinity of at least about 10⁵ M⁻¹and binds to that antigen with an affinity that is higher, for exampleat least two-fold greater, than its binding affinity for an irrelevantantigen (e.g., BSA, casein). The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen.” As used herein, the term “minimal binding”refers to an antibody that does not bind to and/or exhibits low affinityto a specified antigen. Typically, an antibody having minimal binding toan antigen binds to that antigen with an affinity that is lower thanabout 10² M⁻¹ and does not bind to a predetermined antigen with higheraffinity than it binds to an irrelevant antigen.

When used herein for an antibody such as an IgG antibody, the term “highaffinity” refers to a binding affinity of at least about 10⁷ M⁻¹, in atleast one embodiment at least about 10⁸ M⁻¹, in some embodiments atleast about 10⁹ M⁻¹, about 10¹⁰ M⁻¹, about 10¹¹ M⁻¹ or greater, e.g., upto about 10¹³ M⁻¹ or greater. However, “high affinity” binding can varyfor other antibody isotypes. For example, “high affinity” binding for anIgM isotype refers to a binding affinity of at least about 10⁷ M⁻¹.

The term “isotype” as used herein refers to the antibody class (e.g.,IgM or IgG1) that is encoded by heavy chain constant region genes.

The terms “Complementarity-determining region” or “CDR” as used hereinrefers to one of three hypervariable regions within the variable regionof the heavy chain or the variable region of the light chain of anantibody molecule that form the N-terminal antigen-binding surface thatis complementary to the three-dimensional structure of the boundantigen. Proceeding from the N-terminus of a heavy or light chain, thesecomplementarity-determining regions are denoted as “CDR1,” “CDR2,” and“CDR3,” respectively [Wu T T, Kabat E A, Bilofsky H, Proc Natl Acad SciUSA. 1975 December; 72(12):5107 and Wu T T, Kabat E A, J Exp Med. 1970Aug. 1; 132(2):211]. CDRs are involved in antigen-antibody binding, andthe CDR3 comprises a unique region specific for antigen-antibodybinding. An antigen-binding site, therefore, can include six CDRs,comprising the CDR regions from each of a heavy and a light chain Vregion. The term “epitope” refers to the area or region of an antigen towhich an antibody specifically binds or interacts, which in someembodiments indicates where the antigen is in physical contact with theantibody. Conversely, the term “paratope” refers to the area or regionof the antibody on which the antigen specifically binds. Epitopescharacterized by competition binding are said to be overlapping if thebinding of the corresponding antibodies are mutually exclusive, i.e.binding of one antibody excludes simultaneous binding of anotherantibody. The epitopes are said to be separate (unique) if the antigenis able to accommodate binding of both corresponding antibodiessimultaneously.

The term “competing antibodies” as used herein refers to antibodies thatbind to about the same, substantially the same, essentially the same, oreven the same epitope as an antibody against ATβH as described herein.Competing antibodies include antibodies with overlapping epitopespecificities. Competing antibodies are thus able to effectively competewith an antibody as described herein for binding to ATβH. In someembodiments, the competing antibody can bind to the same epitope as theantibody described herein. Alternatively viewed, the competing antibodyhas the same epitope specificity as the antibody described herein.

The term “conservative substitutions” as used herein refers tomodifications of a polypeptide that involve the substitution of one ormore amino acids for amino acids having similar biochemical propertiesthat do not result in loss of a biological or biochemical function ofthe polypeptide. A conservative amino acid substitution is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, value, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Antibodies of the present disclosure can have one or moreconservative amino acid substitutions yet retain antigen bindingactivity.

For nucleic acids and polypeptides, the term “substantial homology” asused herein indicates that two nucleic acids or two polypeptides, ordesignated sequences thereof, when optimally aligned and compared, areidentical, with appropriate nucleotide or amino acid insertions ordeletions, in at least about 80% of the nucleotides or amino acids,usually at least about 85%, in some embodiments about 90%, about 91%,about 92%, about 93%, about 94%, or about 95%, in at least oneembodiment at least about 96%, about 97%, about 98%, about 99%, about99.1%, about 99.2%, about 99.3%, about 99.4%, or about 99.5% of thenucleotides or amino acids. Alternatively, substantial homology fornucleic acids exists when the segments will hybridize under selectivehybridization conditions to the complement of the strand. Also includedare nucleic acid sequences and polypeptide sequences having substantialhomology to the specific nucleic acid sequences and amino acid sequencesrecited herein. The percent identity between two sequences is a functionof the number of identical positions shared by the sequences (i.e., %homology=# of identical positions/total # of positions×100), taking intoaccount the number of gaps, and the length of each gap, that need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, such as withoutlimitation the AlignX™ module of VectorNTI™ (Invitrogen Corp., Carlsbad,Calif.). For AlignX™, the default parameters of multiple alignment are:gap opening penalty: 10; gap extension penalty: 0.05; gap separationpenalty range: 8; % identity for alignment delay: 40.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, such as without limitationthe AlignX™ module of VectorNTI™ (Invitrogen Corp., Carlsbad, Calif.).For AlignX™, the default parameters of multiple alignment are: gapopening penalty: 10; gap extension penalty: 0.05; gap separation penaltyrange: 8; % identity for alignment delay: 40. (further details found athttp://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEACommunities/Vector-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).

Another method for determining the an overall match between a querysequence (a sequence of the present disclosure) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe CLUSTALW computer program (Thompson et al., Nucleic Acids Research,1994, 2(22): 4673-4680), which is based on the algorithm of Higgins etal., Computer Applications in the Biosciences (CABIOS), 1992, 8(2):189-191. In a sequence alignment the query and subject sequences areboth DNA sequences. The result of said global sequence alignment is inpercent identity. Parameters that can be used in a CLUSTALW alignment ofDNA sequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty=10, Gap Extension Penalty=0.1. For multiple alignments, thefollowing CLUSTALW parameters can be used: Gap Opening Penalty=10, GapExtension Parameter=0.05; Gap Separation Penalty Range=8; % Identity forAlignment Delay=40.

The nucleic acids can be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components with which it is normally associated in thenatural environment. To isolate a nucleic acid, standard techniques suchas the following can be used: alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art.

Monoclonal Antibodies Against ATβH

Bleeding disorders where homeostasis is deregulated in hemophilia or intrauma patients where the wound results in a temporary loss ofhemostasis, can be treated by AT inhibitors. Antibodies, antigen-bindingfragments thereof, and other AT-specific protein scaffolds can be usedto provide targeting specificity to inhibit a subset of AT proteinfunctions while preserving the rest. Given the at least 10-folddifference in plasma concentration of ATβ (<12 ug/ml) versus ATα (120ug/ml), increased specificity of any potential ATβ inhibitortherapeutics is helpful to block ATβ function in the presence of a highcirculating excess of ATα. ATβ specific antibodies that block theanti-coagulant function of ATβ can be used as therapeutics for patientswith bleeding disorders. Examples of bleeding disorders includehemophilia, hemophilia patients with inhibitors, trauma-inducedcoagulopathy, severe bleeding patients during sepsis treatment by AT,bleeding resulting from elective surgery such as transplantation,cardiac surgery, orthopedic surgery, and excessive bleeding fromMenorrhagia. Anti-ATβH antibodies having long circulating half-live canbe useful in treating chronic diseases like hemophilia. ATβH antibodyfragments or ATβH-binding protein scaffolds with shorter half-lives canbe more effective for acute use (e.g. therapeutic use in trauma),ATβH-binding antibodies were identified by panning and screening humanantibody libraries against human ATβ in complex with heparin. Theidentified antibodies exhibited binding to human ATβH. The heavy chainvariable region and light chain variable region of each monoclonalantibody isolated was sequenced and its CDR regions were identified. Thesequence identifier numbers (“SEQ ID NO”) that correspond to the heavyand light chain variable regions of the ATβH-specific monoclonalantibodies arc summarized in Table 1A.

TABLE 1A Human anti-ATβH (heparin complexed ATβ) antibodies SEQ HeavyChain Variable SEQ Clone Light Chain variable Region ID Region IDTPP2009 AQSVLTQDPAVSVALGQTVRIT No. 1 EVQLLESGGGLVQPG No. 2CQGDSLRSYYASWYQQKPGQ GSLRLSCAASGFTFS APVLVIYGKNNRPSGIPDRFSGSAYRMGWVRQAPGK SSGNTASLTITGAQAEDEADYY GLEWVSRIYSSGGRTCNSRDSSGNHLVFGGGTKLTV RYADSVKGRFTISRD LGQPKAAPSVTLFPPSSEELQANSKNTLYLQMNSLR NKATLVCLISDFYPGAVTVAW AEDTAVYYCAREKAKADGSPVKAGVETTKPSKQSN SDLSGSFSEALDYWG NKYAASSYLSLTPEQWKSHRS QGTLVTVSSYSCQVTHEGSTVEKTVAPAECS TPP2015 AQDIQMTQSPGTLSLSPGERAT No. 3EVQLLESGGGLVQPG No. 4 LSCRASQSVSSSYLAWYQQKP GSLRLSCAASGFTFSGQAPRLLIYGASSRATGIPDRFS KYKMDWVRQAPGK GSGSGTDFTLTISRLEPEDFAVYGLEWVSRIGPSGGKT YCQQYGSSRTFGQGTKVEIRRT MYADSVKGRFTISRDVAAPSVFIFPPSDEQLKSGTASV NSKNTLYLQMNSLR VCLLNNFYPREAKVQWKVDNAEDTAVYYCAREKA ALQSGNSQESVTEQDSKDSTYS SDLSGTYSEALDYWLSSTLTLSKADYEKHKVYACE GQGTLVTVSS VTH QGLSSPVTKS FNRGEC TPP2016AQDIQMTQSPATLSVSPGERAT No. 5 EVQLLESGGGLVQPG No. 6 LSCRASQNINRNLAWYQQKPGGSLRLSCAASGFTFS RAPRLLIHTASTRAPGVPVRITG KYRMDWVRQAPGKSGSGTEFTLTISSLEPEDFAVYF GLEWVSRIGPSGGKT CQQYASPPRTFGQGTKVEIKRTTYADSVKGRFTISRD VAAPSVFIFPPSDEQLKSGTASV NSKNTLYLQMNSLRVCLLNNFYPREAKVQWKVDN AEDTAVYYCAREKT ALQSGNSQESVTEQDSKDSTYSSDLSGSYSBALDYW LSST LTLSKADYEK GQGTL VTVSS HKVYACEVTH QGLSSPVTKS FNRGECTPP2019 AQDIQMTQSPATTLSLSPGBRAT No. 7 EVQLLESGGG No. 8LSCRASQRVSSSYLTWYQQKP LVQPGGSLRL GQAPRLLIYGASSRATGIPDRFS SCAASGFTFSGSGSGTDFTLTISRLEPEDFAVY RYAMYWVRQA YCQQYDSTPPLTFGGGTKVEIK PGKGLEWVSRRTVAAPSVFIFPPSDEQLKSGTA ISPSGGKTHY SVVCLLNNFYPREAKVQWKVD ADSVKGRFTINALQSGNSQESVTEQDSKDST SRDNSKNTLY YSLS LQMNSLRAED STLTLSKADYEKHKVYACEVTTAVYYCARLS HQGLSSPVTKSFNRGEC QTGYYPHYHY YGMDVWGQGT TVTVSS TPP2803SSELTQDPAVSVALGQTVRITC No. 9 EVQLLESGGGLVQPG No. 10QGDSLRSYYASWYQQKPGQAP GSLRLSCAASGFTFSS VLVTYGKNNRPSGIPDRFSGSSSYRMSWVRQAPGKGL GNTASLTITGAQAEDEADYYC EWVSRIYSSGGRTRYNSRDSSGNHLVFGGGTKLTVL ADSVKGRFTISRDNS GQPKAAPSVTLFPPSSEELQANKNTLYLQMNSLRAE KATLVCLISDFYPGAVTVAWK DTAVYYCAREKASDADGSPVKAGVETTKPSKQSNN LSGSFSEALDYWGQ KYAASSYLSLTPEQWKSHRSYS GTLVTVSSCQVTHEGSTVEKTVAPAECS

In at least some possible embodiments, an isolated monoclonal antibodybinds to human and inhibits anticoagulant activity, wherein the antibodycomprises a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8,and 10.

In at least some possible embodiments, an isolated monoclonal antibodybinds to human ATβH and inhibits anticoagulant activity, wherein theantibody comprises a light chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5,7, and 9.

In at least some possible embodiments an isolated monoclonal antibodybinds to human ATβH and inhibits anticoagulant activity, wherein theantibody comprises a heavy chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6,8, and 10 and a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7,and 9.

In at least some possible embodiments, the antibody comprises heavy andlight chain variable regions comprising:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence of SEQ ID NO: 2, and a light chain variable region        comprising an amino acid sequence of SEQ ID NO: 1;    -   (b) a heavy chain variable region comprising an amino acid        sequence of SEQ ID NO: 4, and a light chain variable region        comprising an amino acid sequence of SEQ ID NO: 3;    -   (c) a heavy chain variable region comprising an amino acid        sequence of SEQ ID NO: 6, and a light chain variable region        comprising an amino acid sequence of SEQ ID NO: 5; or    -   (d) a heavy chain variable region comprising an amino acid        sequence of SEQ ID NO: 8, and a light chain variable region        comprising an amino acid sequence of SEQ ID NO: 7; or    -   (e) a heavy chain variable region comprising an amino acid        sequence of SEQ ID NO: 10, and a light chain variable region        comprising an amino acid sequence of SEQ ID NO: 9.

Table 1B shows heavy and light chain amino acid sequences for humanizedIgG mAbs.

TABLE 1B Heavy and Light Chain Amino Acid Sequences for humanized IgGmAbs. TPP2009|hIgG|Light_ChainAQSVLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEADYYCNSRDSSGNHLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPAECS SEQID NO: 51 TPP2009|hIgG|Heavy chainEVQLLESGGGLVQPGGSLRLSCAASGPTFSAYRMGWVRQAPGKGLEWVSRIYSSGGRTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKASDLSGSFSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYPPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO:52TPP-2015|hIgGlight_chainAQDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRTFGQGTKVEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQID NO: 53 TPP-2015|hIgG|heavy_chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMDWVRQAPGKGLEWVSRIGPSGGKTMYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKASDLSGTYSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGrAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSG SEQ IDNO: 54TPP-2016|hIgG|light_chain, KappaAQDIQMTQSPATLSVSPGERATLSCRASQNINRNLAWYQQKPGRAPRLLIHTASTRAPGVPVRITGSGSGTEFTLTISSLEPEDFAVYFCQQYASPPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAEQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQID NO: 55 TPP-2016|hIgG|heavy_chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSKYRMDWVRQAPGKGLEWVSRIGPSGGKTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKTSDLSGSYSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVWSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNYNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVYDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPBNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG SEQ IDNO: 56TPP-2019|hIgG|light_chain, KappaAQDIQMTQSPATLSLSPGERATLSCRASQRVSSSYLTWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSTPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO: 57 TPP-2019|hIgG|heavy_chaninEVQLLESGGGLVQPGGSLRLSCAASGFTSRYAMYWVRQAPGKGLEWVSRISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLSQTGYYPHYHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO: 58

Table 1C—shows TPP2803 IgG2, Germlined and converted to IgG2. TPP2803IgG2 light chain G2, Lambda, amino acid sequence shown in Table 1C isSEQ ID NO:59 and TPP2803 heavy chain amino acid sequence shown in Table1C is SEQ ID NO: 60.

TABLE 1C TPP2803 IgG2, Germlined and converted toIgG2 >TPP-2803|hIgG2|light_chain, lambdaSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPAECS SEQ IDNO: 59 >TPP-2803|hIgG2|heavy_chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYRMSWVRQAPGKGLEWVSRIYSSGGRTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKASDLSGSFSEALDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVFHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G SEQ ID NO: 60

Table 2A provides a summary of the SEQ ID NOS: for the CDR regions(“CDR1,” “CDR2,” and “CDR3”) of heavy and light chains of monoclonalantibodies that bind to human ATβH.

TABLE 2A Sequence Identifiers for CDR Regions of Human Anti-ATβHAntibodies Light Chain Heavy Chain Variable Region Variable RegionClones CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 TPP2009 21 26 31 36 41 46 TPP201522 27 32 37 42 47 TPP2016 23 28 33 38 43 48 TPP2019 24 29 34 39 44 49TPP2803 25 30 35 40 45 50

Table 2B provides sequences of the SEQ ID NOS: for the CDR regions(“CDR1,” “CDR2,” and “CDR3”) of heavy and light chains of monoclonalantibodies that bind to human ATβH.

TABLE 2B Sequences for CDR Regions of Human Anti-ATβH AntibodiesSequence- Clone CDR Identifier Amino Acid Sequence TPP2009 LCDR1 SEQ IQNO: 21 QGDSLRSYYAS TPP2015 LCDR1 SEQ ID NO: 22 RASQSVSSSYLA TPP2016LCDR1 SEQ ID NO: 23 RASQNINRNLA TPP2019 LCDR1 SEQ ID NO: 24 RASQRVSSSYLTTPP2803 LCDR1 SEQ ID NO: 25 QGDSLRSYYAS TPP2009 LCDR2 SEQ ID NO: 26GKNNRPS TPP2015 LCDR2 SEQ ID NO: 27 GASSRAT TPP2016 LCDR2 SEQ ID NO: 28TASTRAP TPP2019 LCDR2 SEQ ID NO: 29 GASSRAT TPP2803 LCDR2 SEQ ID NO: 30GKNNRPS TPP2009 LCDR3 SEQ ID NO: 31 NSRDSSGNHLV TPP2015 LCDR3 SEQ ID NO:32 QQYGSSRT TPP2016 LCDR3 SEQ ID NO: 33 QQYASPPRT TPP2019 LCDR3 SEQ IDNO: 34 QQYDSTPPLT TPP2803 LCDR3 SEQ ID NO: 35 NSRDSSGNHLV TPP2009 HCDR1SEQ ID NO: 36 AYRMG TPP2015 HCDR1 SEQ ID NO: 37 KYKMD TPP2016 HCDR1 SEQID NO: 38 KYRMD TPP2019 HCDR1 SEQ ID NO: 39 RYAMY TPP2803 HCDR1 SEQ IDNO: 40 SYRMS TPP2009 HCDR2 SEQ ID NO: 41 RIYSSGGRTRYADSVKG TPP2015 HCDR2SEQ ID NO: 42 RIGPSGGKTM YADSVKG TPP2016 HCDR2 SEQ ID NO: 43 RIGPSGGKTTYADSVKG TPP2019 HCDR2 SEQ ID NO: 44 RISPSGGKTH YADSVKG TPP2803 HCDR2 SEQID NO: 45 RIYSSGGRTR YADSVKG TPP2009 HCDR3 SEQ ID NO: 46AREKASDLSGSFSEALDY TPP2015 HCDR3 SEQ ID NO: 47 AREKASDLSG TYSEALDYTPP2016 HCDR3 SEQ ID NO: 48 AREKTSDLSG SYSEALDY TPP2019 HCDR3 SEQ ID NO:49 ARLSQTGYYP HYHYYGMDV TPP2803 HCDR3 SEQ ID NO: 50 AREKASDLSG SFSEALDY

In at least some possible embodiments, an isolated monoclonal antibodyis provided that binds to human ATβH, wherein the antibody comprises aCDR3 comprising an amino acid sequence of any one of SEQ ID NOS: 46-50.These CDR3s are from a heavy chain of the antibodies identified duringpanning and screening.

In a further embodiment, this antibody further comprises: (a) a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 36-40; (b) a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 41-45; or (c) both a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 36-40 and a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 41-45.

In at least some possible embodiments, antibodies share a CDR3 from oneof the light chains of the antibodies identified during panning andscreening. Thus, also provided is an isolated monoclonal antibody,wherein said antibody binds to ATβH and inhibits anticoagulant activity,wherein said antibody comprises a CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS: 31-35. In furtherembodiments, the antibody further comprises (a) a CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOS:21-25,(b) a CDR2 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 26-30, or (c) both a CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOS:21-25 and a CDR2 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 26-30.

In at least some possible embodiments, the antibody comprises a CDR3from a heavy chain and a light chain of the antibodies identified fromscreening and panning. Provided is an isolated monoclonal antibody,wherein said antibody binds to ATβH and inhibits anticoagulant activity,wherein said antibody comprises a CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS: 46-50 and a CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 31-35. In a further embodiment, the antibody furthercomprises: (a) a CDR1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 36-40; (b) a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOS:41-45; (c) a CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 21-25; and/or (d) a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:26-30.

In some embodiments, the antibody comprises heavy and light chainvariable regions comprising:

-   -   (a) a light chain variable region comprising an amino acid        sequence comprising SEQ ID NOS: 21, 26, and 31 and a heavy chain        variable region comprising an amino acid sequence comprising SEQ        ID NOS: 36, 41, and 46;    -   (b) a light chain variable region comprising an amino acid        sequence comprising SEQ ID NOS: 22, 27, and 32 and a heavy chain        variable region comprising an amino acid sequence comprising SEQ        ID NOS: 37, 42, and 47;    -   (c) a light chain variable region comprising an amino acid        sequence comprising SEQ ID NOS: 23, 28, and 33 and a heavy chain        variable region comprising an amino acid sequence comprising SEQ        ID NOS: 38, 43, and 48;    -   (d) a light chain variable region comprising an amino acid        sequence comprising SEQ ID NOS: 24, 29, and 34 and a heavy chain        variable region comprising an amino acid sequence comprising SEQ        ID NOS: 39, 44, and 49;    -   (e) a light chain variable region comprising an amino acid        sequence comprising SEQ ID NOS: 25, 30, and 35 and a heavy chain        variable region comprising an amino acid sequence comprising SEQ        ID NOS: 40, 45, and 50.

Also provided is an isolated monoclonal antibody that binds to AtβH andinhibits anticoagulant activity, wherein said antibody comprises anamino acid sequence having at least about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 99.5% identity to an amino acid sequenceselected from the group consisting of the amino acid sequences set forthin SEQ ID NOS: 1-10.

The antibody can be species specific or can cross react with multiplespecies. In some embodiments, the antibody can specifically react orcross react with ATβH of human, mouse, rat, rabbit, guinea pig, monkey,pig, dog, cat or other mammalian species.

The antibody can be of any of the various classes of antibodies, such aswithout limitation an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1,an IgA2, a secretory IgA, and IgD, and an IgE antibody.

In one embodiment, provided is an isolated fully human monoclonalantibody to human ATIII.

Optimized Variants of Anti-ATβH Antibodies

In some embodiments, the antibodies can be panned, screened andoptimized, for example to increase affinity to ATβH, to further decreaseany affinity to ATα, to improve cross-reactivity to different species,or to improve blocking activity of ATβH. Such optimization can beperformed for example by utilizing site saturation mutagenesis of theCDRs or amino acid residues in close proximity to the CDRs, i.e. about 3or 4 residues adjacent to the CDRs, of the antibodies.

Also provided are monoclonal antibodies that may have increased or highaffinity to ATβH. In some embodiments, the anti-ATβH antibodies may havea binding affinity of at least about 10⁸M⁻¹, in some other embodimentsmay have at least about 10⁹M⁻¹, about 10¹⁰M⁻¹, about 10¹¹M⁻¹ or greater,e.g., up to about 10¹³M⁻¹ or greater.

In some embodiments, additional amino acid modifications can beintroduced to reduce divergence from the germ line sequence. In otherembodiments, amino acid modifications can be introduced to facilitateantibody production for large scale production processes.

In some embodiments, provided are isolated anti-ATβH monoclonalantibodies that specifically bind to human ATβ, which antibodies maycomprise one or more amino acid modifications. In some embodiments, theantibody may comprise about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, or about 20or more modifications.

Epitopes

Also provided is an isolated monoclonal antibody that can bind to apredicted epitope of human ATβH, wherein the epitope comprises one ormore of residues from human ATβH as shown in FIG. 11.

In some embodiments, the epitope comprises the N135 site of human ATβH.In other embodiments, the site can comprise part of the amino acidresidue sequence of RCL loop of human ATβH.

Also provided are antibodies that can compete with any of the antibodiesdescribed herein for binding to human ATβH. For example, such acompeting antibody can bind to one or more epitopes described above.

Nucleic Acids, Vectors and Host Cells

Also provided are isolated nucleic acid molecules encoding any of themonoclonal antibodies described herein. Thus, provided is an isolatednucleic acid molecule encoding an antibody that binds to human ATβH.Table 3 shows the nucleotide sequences of some anti-ATβH antibodies.

TABLE 3 Nucleotide sequence of anti-ATβH antibodies. Light Chain HeavyChain TPP2009 GCACAGAGCGTCTTG GAAGTTCAATTGTTAGAGTCTGGTGG ACTCAGGACCCTGCTCGGTCTTGTTCAGCCTGGTGGTTCTTT GTGTCTGTGGCCTTG ACGTCTTTCTTGCGCTGCTTCCGGATTGGACAGACAGTCAG CACTTTCTCTGCTTACCGTATGGGTTG GATCACATGCCAAGGGGTTCGCCAAGCTCCTGGTAAAGGTT AGACAGCCTCAGAA TGGAGTGGGTTTCTCGTATCTATTCTTGCTATTATGCAAGCT CTGGTGGCCGTACTCGTTATGCTGACT GGTACCAGCAGAAGCCGTTAAAGGTCGCTTCACTATCTCTA CCAGGACAGGCCCCT GAGACAACTCTAAGAATACTCTCTACGTACTTGTCATCTAT TTGCAGATGAACAGCTTAAGGGCTGA GGTAAAAACAACCGGGACACGGCCGTGTATTACTGTGCGA GCCCTCAGGGATCCC GAGAGAAAGCGTCGGATCTATCGGGGAGACCGATTCTCTGG AGTTTTTCTGAGGCCCTTGACTACTGG CTCCAGCTCAGGAAAGGCCAGGGAACCCTGGTCACCGTCTC CACAGCTTCCTTGAC AAGCGCCTCCACCAAGGGCCCATCGGCATCACTGGGGCTCA TCTTCCCGCTAGCACCCAGCAGCAAG GGCGGAAGATGAGGAGCACCAGCGGCGGAACAGCCGCCCT CTGACTATTACTGTA GGGCTGCCTGGTGAAAGACTACTTCCACTCCCGGGACAGCA CCGAGCCCGTGACCGTGTCCTGGAAC GTGGTAACCATCTGGTCTGGCGCCCTGACCAGCGGAGTGCA TATTCGGCGGAGGGA TACCTTCCCCGCCGTGCTGCAGAGCACCAAGCTGACCGTCC GCGGCCTGTACAGCCTGAGCAGCGTG TAGGTCAGCCCAAGGGTGACAGTGCCCAGCAGCAGCCTGGG CTGCCCCCTCGGTCA AACCCAGACCTACATCTGCAACGTGACTCTGTTCCCGCCCT ACCACAAGCCCAGCAACACCAAGGTG CCTCTGAGGAGCTTCGACAAGAAGGTGGAACCCAAGAGCT AAGCCAACAAGGCC GCGACAAGACCCACACCTGTCCCCCCACACTAGTGTGTCTG TGCCCTGCCCCTGAACTGCTGGGCGG ATCAGTGACTTCTACACCCAGCGTGTTCCTGTTCCCCCCAAA CCGGGAGCTGTGACA GCCCAAGGACACCCTGATGATCAGCCGTGGCCTGGAAGGCA GGACCCCCGAAGTGACCTGCGTGGTG GATGGCAGCCCCGTCGTGGACGTGTCCCACGAGGACCCAGA AAGGCGGGAGTGGA AGTGAAGTTTAATTGGTACGTGGACGGACCACCAAACCCTC GCGTGGAAGTGCATAACGCCAAGACC CAAACAGAGCAACAAAGCCCAGAGAGGAACAGTACAACA ACAAGTACGCGGCCA GCACCTACCGGGTGGTGTCCGTGCTGGCAGCTACCTGAGCC ACCGTGCTGCACCAGGACTGGCTGAA TGACGCCCGAGCAGTCGGCAAAGAGTACAAGTGCAAGGTCT GGAAGTCCCACAGA CCAACAAGGCCCTGCCTGCCCCCATCAGCTACAGCTGCCAG GAGAAAACCATCAGCAAGGCCAAGG GTCACGCATGAAGGGGCCAGCCCCGCGAGCCTCAGGTGTAC AGCACCGTGGAGAA ACACTGCCCCCCAGCCGGGATGAGCTGACAGTGGCCCCTGC GACCAAGAACCAGGTGTCCCTGACCT AGAATGCTCT (SEQGTCTGGTGAAAGGCTTCTACCCCAGC ID NO: 11) GATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAATTACAAGA CCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTG ACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGA TGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGAGCCC CGGC (SEQ ID NO: 12) TPP2015 GCACAAGACATCCAGGAAGTTCAATTGTTAGAGTCTGGTGG ATGACCCAGTCTCCA CGGTCTTGTTCAGCCTGGTGGTTCTTTGGCACCCTGTCTTTG ACGTCTTTCTTGCGCTGCTTCCGGATT TCTCCAGGGGAAAGACACTTTCTCTAAGTACAAGATGGATTG GCCACCCTCTCCTGC GGTTCGCCAAGCTCCTGGTAAAGGTTAGGGCCAGTCAGAGT TGGAGTGGGTTTCTCGTATCGGTCCTT GTTAGCAGCAGCTACCTGGTGGCAAGACTATGTATGCTGAC TTAGCCTGGTACCAG TCCGTTAAAGGTCGCTTCACTATCTCTCAGAAACCTGGCCAG AGAGACAACTCTAAGAATACTCTCTA GCTCCCAGGCTCCTCCTTGCAGATGAACAGCTTAAGGGCTG ATCTATGGTGCATCC AGGACACGGCCGTGTATTACTGTGCGAGCAGGGCCACTGGC AGAGAGAAAGCGTCGGATCTATCGGG ATCCCAGACAGGTTCGACTTATTCTGAGGCCCTTGACTACTG AGTGGCAGTGGGTCT GGGCCAGGGAACCCTGGTCACCGTCTGGGACAGACTTCACT CAAGCGCCTCCACCAAGGGCCCATCG CTCACCATCAGCAGAGTCTTCCCGCTAGCACCCAGCAGCAA CGGAGCCTGAAGATT GAGCACCAGCGGCGGAACAGCCGCCCTTGCAGTGTATTACT TGGGCTGCCTGGTGAAAGACTACTTC GTCAGCAGTATGGTACCCGAGCCCGTGACCGTGTCCTGGAA GCTCAACGTTCGGCC CTCTGGCGCCCTGACCAGCGGAGTGCAAGGGACCAAGGTG ATACCTTCCCCGCCGTGCTGCAGAGC GAAATCAGACGAACTAGCGGCCTGTACAGCCTGAGCAGCGT GTGGCTGCAATCTGT GGTGACAGTGCCCAGCAGCAGCCTGGCTTCATCTTCCCGCC GAACCCAGACCTACATCTGCAACGTG ATCTGATGAGCAGTTAACCACAAGCCCAGCAACACCAAGGT GAAATCTGGAACTGC GGACAAGAAGGTGGAACCCAAGAGCCTCTGTTGTGTGCCT TGCGACAAGACCCACACCTGTCCCCC GCTGAATAACTTCTACTGCCCTGCCCCTGAACTGCTGGGCG TCCCAGAGAGGCCAA GACCCAGCGTGTTCCTGTTCCCCCCAAAGTACAGTGGAAGGT AGCCCAAGGACACCCTGATGATCAGC GGATAACGCCCTCCACGGACCCCCGAAGTGACCTGCGTGGT ATCGGGTAACTCCCA GGTGGACGTGTCCCACGAGGACCCAGGGAGAGTGTCACAG AAGTGAAGTTTAATTGGTACGTGGAC AGCAGGACAGCAAGGGCGTGGAAGTGCATAACGCCAAGAC GACAGCACCTACAGC CAAGCCCAGAGAGGAACAGTACAACCTCAGCAGCACCCTG AGCACCTACCGGGTGGTGTCCGTGCT ACGCTGAGCAAAGCGACCGTGCTGCACCAGGACTGGCTGA AGACTACGAGAAAC ACGGCAAAGAGTACAAGTGCAAGGTCACAAAGTCTACGCCT TCCAACAAGGCCCTGCCTGCCCCCAT GCGAAGTCACCCATCCGAGAAAACCATCAGCAAGGCCAAG AGGGCCTGAGCTCGC GGCCAGCCCCGCGAGCCTCAGGTGTACCGTCACAAAGAGCT CACACTGCCCCCCAGCCGGGATGAGC TCAACAGGGGAGAGTGACCAAGAACCAGGTGTCCCTGACC TGT (SEQ ID NO: 13)TGTCTGGTGAAAGGCTTCTACCCCAG CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAATTACAAG ACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCT GACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTG ATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGC (SEQ ID NO: 14) TPP2016 GCACAAGACATCCAGGAAGTTCAATTGTTAGAGTCTGGTGG ATGACCCAGTCTCCA CGGTCTTGTTCAGCCTGGTGGTTCTTTGCCACCCTGTCTGTG ACGTCTTTCTTGCGCTGCTTCCGGATT TCTCCAGGGGAAAGACACTTTCTCTAAGTACCGTATGGATTG GCCACCCTCTCCTGC GGTTCGCCAAGCTCCTGGTAAAGGTTAGGGCCAGTCAGAAT TGGAGTGGGTTTCTCGTATCGGTCCTT ATTAATAGAAACTTGCTGGTGGCAAGACTACTTATGCTGAC GCCTGGTACCAGCAG TCCGTTAAAGGTCGCTTCACTATCTCTAAGCCTGGCCGGGCT AGAGACAACTCTAAGAATACTCTCTA CCCAGACTCCTCATCCTTGCAGATGAACAGCTTAAGGGCTG CATACCGCATCCACT AGGACACGGCCGTGTATTACTGTGCGAGGGCCCCTGGTGTC AGAGAGAAAACGTCGGATCTATCGGG CCAGTCAGGATCACTGAGTTATTCTGAGGCCCTTGACTACTG GGCAGTGGGTCTGGA GGGCCAGGGAACCCTGGTCACCGTCTACAGAGTTCACTCTC CAAGCGCCTCCACCAAGGGCCCATCG ACCATCAGCAGCCTGGTCTTCCCGCTAGCACCCAGCAGCAA GAACCTGAAGATTTT GAGCACCAGCGGCGGAACAGCCGCCCGCAGTGTATTTCTGT TGGGCTGCCTGGTGAAAGACTACTTC CAGCAGTATGCTAGCCCCGAGCCCGTGACCGTGTCCTGGAA CCACCTCGGACGTTC CTCTGGCGCCCTGACCAGCGGAGTGCGGCCAAGGGACCAA ATACCTTCCCCGCCGTGCTGCAGAGC GGTGGAAATCAAGCAGCGGCCTGTACAGCCTGAGCAGCGT GAACTGTGGCTGCAC GGTGACAGTGCCCAGCAGCAGCCTGGCATCTGTCTTCATCTT GAACCCAGACCTACATCTGCAACGTG CCCGCCATCTGATGAAACCACAAGCCCAGCAACACCAAGGT GCAGTTGAAATCTGG GGACAAGAAGGTGGAACCCAAGAGCAACTGCCTCTGTTGT TGCGACAAGACCCACACCTGTCCCCC GTGCCTGCTGAATAACTGCCCTGCCCCTGAACTGCTGGGCG CTTCTATCCCAGAGA GACCCAGCGTGTTCCTGTTCCCCCCAAGGCCAAAGTACAGTG AGCCCAAGGACACCCTGATGATCAGC GAAGGTGGATAACGCGGACCCCCGAAGTGACCTGCGTGGT CCCTCCAATCGGGTA GGTGGACGTGTCCCACGAGGACCCAGACTCCCAGGAGAGTG AAGTGAAGTTTAATTGGTACGTGGAC TCACAGAGCAGGACGGCGTGGAAGTGCATAACGCCAAGAC AGCAAGGACAGCAC CAAGCCCAGAGAGGAACAGTACAACCTACAGCCTCAGCAG AGCACCTACCGGGTGGTGTCCGTGCT CACCCTGACGCTGAGGACCGTGCTGCACCAGGACTGGCTGA CAAAGCAGACTACG ACGGCAAAGAGTACAAGTGCAAGGTCAGAAACACAAAGTCT TCCAACAAGGCCCTGCCTGCCCCCAT ACGCCTGCGAAGTCACGAGAAAACCATCAGCAAGGCCAAG CCCATCAGGGCCTGA GGCCAGCCCCGCGAGCCTCAGGTGTAGCTCGCCCGTCACAA CACACTGCCCCCCAGCCGGGATGAGC AGAGCTGACCAAGAACCAGGTGTCCCTGACC TTCAACAGGGGAGA TGTCTGGTGAAAGGCTTCTACCCCAGGTGT (SEQ ID NO: 15) CGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAATTACAAG ACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCT GACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTG ATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGC (SEQ ID NO: 16) TPP2019 GCACAAGACATCCAGGAAGTTCAATTGTTAGAGTCTGGTGG ATGACCCAGTCTCCA CGGTCTTGTTCAGCCTGGTGGTTCTTTGCCACCCTGTCTTTG ACGTCTTTCTTGCGCTGCTTCCGGATT TCTCCAGGGGAAAGACACTTTCTCTCGTTACGCTATGTATTG GCCACCCTCTCCTGC GGTTCGCCAAGCTCCTGGTAAAGGTTAGGGCCAGTCAGCGT TGGAGTGGGTTTCTCGTATCTCTCCTT GTTAGCAGCAGCTACCTGGTGGCAAGACTCATTATGCTGAC TTAACCTGGTACCAG TCCGTTAAAGGTCGCTTCACTATCTCTCAGAAACCTGGCCAG AGAGACAACTCTAAGAATACTCTCTA GCTCCCAGGCTCCTCCTTGCAGATGAACAGCTTAAGGGCTG ATCTATGGTGCATCC AGGACACGGCCGTGTATTACTGTGCGAGCAGGGCCACTGGC AGACTGTCTCAAACTGGTTATTACCCT ATCCCAGACAGGTTCCACTACCACTACTACGGTATGGACGT AGTGGCAGTGGGTCT CTGGGGCCAAGGGACCACGGTCACCGGGGACAGACTTCACT TCTCAAGCGCCTCCACCAAGGGCCCA CTCACCATCAGCAGATCGGTCTTCCCGCTAGCACCCAGCAG CTGGAGCCTGAAGAT CAAGAGCACCAGCGGCGGAACAGCCTTTGCAGTTTATTACT GCCCTGGGCTGCCTGGTGAAAGACTA GTCAGCAGTATGATACTTCCCCGAGCCCGTGACCGTGTCCTG GTACGCCTCCGCTCA GAACTCTGGCGCCCTGACCAGCGGAGCCTTCGGCGGAGGGA TGCATACCTTCCCCGCCGTGCTGCAGA CCAAGGTGGAGATCAGCAGCGGCCTGTACAGCCTGAGCAGC AACGAACTGTGGCTG GTGGTGACAGTGCCCAGCAGCAGCCTCACCATCTGTCTTCA GGGAACCCAGACCTACATCTGCAACG TCTTCCCGCCATCTGTGAACCACAAGCCCAGCAACACCAAG ATGAGCAGTTGAAAT GTGGACAAGAAGGTGGAACCCAAGACTGGAACTGCCTCTG GCTGCGACAAGACCCACACCTGTCCC TTGTGTGCCTGCTGACCCTGCCCTGCCCCTGAACTGCTGGGC ATAACTTCTATCCCA GGACCCAGCGTGTTCCTGTTCCCCCCAGAGAGGCCAAAGTA AAGCCCAAGGACACCCTGATGATCAG CAGTGGAAGGTGGATCCGGACCCCCGAAGTGACCTGCGTGG AACGCCCTCCAATCG TGGTGGACGTGTCCCACGAGGACCCAGGTAACTCCCAGGAG GAAGTGAAGTTTAATTGGTACGTGGA AGTGTCACAGAGCAGCGGCGTGGAAGTGCATAACGCCAAGA GACAGCAAGGACAG CCAAGCCCAGAGAGGAACAGTACAACCACCTACAGCCTCAG AGCACCTACCGGGTGGTGTCCGTGCT CAGCACCCTGACGCTGACCGTGCTGCACCAGGACTGGCTGA GAGCAAAGCAGACT ACGGCAAAGAGTACAAGTGCAAGGTCACGAGAAACACAAA TCCAACAAGGCCCTGCCTGCCCCCAT GTCTACGCCTGCGAACGAGAAAACCATCAGCAAGGCCAAG GTCACCCATCAGGGC GGCCAGCCCCGCGAGCCTCAGGTGTACTGAGCFCGCCCGTC CACACTGCCCCCCAGCCGGGATGAGC ACAAAGAGCTTCAACTGACCAAGAACCAGGTGTCCCTGACC AGGGGAGAGTGT TGTCTGGTGAAAGGCTTCTACCCCAG (SEQID NO: 17) CGATATCGCCGTGGAATGGGAGAGCA ACGGCCAGCCCGAGAACAATTACAAGACCACCCCCCCTGTGCTGGACAGCGA CGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGC AGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTA CACCCAGAAGTCCCTGAGCCTGAGCC CCGGC (SEQ ID NO:18) TPP2803 AGCGAATTGACTCAG GAAGTGCAGCTGCTGGAAAGCGGCGG GACCCTGCTGTGTCTAGGCCTGGTGCAGCCTGGCGUATCTC GTGGCCTTGGGACAG TGAGACTCTAGCTGTGCCGCCAGCGGCACAGTCAGGATCACA TTCACCTTCAGCAGCTACAGAATGAG TGCCAAGGAGACAGCTGGGTGCGCCAGGCCCCTGGCAAGG CCTCAGAAGCTATTA GACTGGAATGGGTGTCCCGGATCTACTGCAAGCTGGTACCA AGCAGCGGCGGCAGAACCAGATACGC GCAGAAGCCAGGACCGACAGCGTGAAGGGCCGGTTCACCA AGGCCCCTGTACTTG TCTCCCGGGACAACAGCAAGAACACCTCATCTATGGTAAAA CTGTACCTGCAGATGAACAGCCTGCG ACAACCGGCCCTCAGGGCCGAGGACACCGCCGTGTACTATT GGATCCCAGACCGAT GCGCCAGAGAGAAGGCCAGCGACCTGTCTCTGGCTCCAGCT AGCGGCAGCTTTAGCGAGGCCCTGGA CAGGAAACACAGCTTTTATTGGGGCCAGGGCACACTCGTGA CCTTGACCATCACTG CCGTGTCTAGCGCCAGCACAAAGGGCGGGCTCAGGCGGAA CCCAGCGTGTTCCCTCTGGCCCCTTGT GATGAGGCTGACTATAGCAGAAGCACCAGCGAGTCTACAGC TACTGTAACTCCCGG CGCCCTGGGCTGCCTCGTGAAGGACTGACAGCAGTGGTAAC ACTTTCCCGAGCCCGTGACAGTGTCCT CATCTGGTATTCGGCGGAACTCTGGCGCCCTGACAAGCGGC GGAGGGACCAAGCT GTGCACACCTTTCCAGCCGTGCTGCAGACCGTCCTAGGTCA GAGCAGCGGCCTGTACTCTCTGAGCA GCCCAAGGCTGCCCCGCGTCGTGACTGTGCCCAGCAGCAAC CTCGGTCACTCTGTT TTCGGCACCCAGACCTACACCTGTAACCCGCCCTCCTCTGA CGTGGACCACAAGCCCAGCAACACCA GGAGCTTCAAGCCAAAGGTGGACAAGACCGTGGAACGGAA CAAGGCCACACTAGT GTGCTGCGTGGAATGCCCCCCTTGTCCGTGTCTGATCAGTGA TGCCCCTCCAGTGGCTGGCCCTTCCGT CTTCTACCCGGGAGCGTTCCTGTTCCCCCCAAAGCCCAAGG TGTGACAGTGGCCTG ACACCCTGATGATCAGCCGGACCCCGGAAGGCAGATGGCA AAGTGACCTGCGTGGTGGTGGATGTG GCCCCGTCAAGGCGGTCCCACGAGGACCCCGAGGTGCAGTT GAGTGGAGACCACC CAATTGGTACGTGGACGGCGTGGAAGAAACCCTCCAAACAG TGCACAACGCCAAGACCAAGCCCAGA AGCAACAACAAGTAGAGGAACAGTTCAACAGCACCTTCCG CGCGGCCAGCAGCTA GGTGGTGTCCGTGCTGACCGTGGTGCCCTGAGCCTGACGCC ATCAGGACTGGCTGAACGGCAAAGAG CGAGCAGTGGAAGTCTACAAGTGCAAGGTGTCCAACAAGGG CCACAGAAGCTACAG CCTGCCTGCCCCCATCGAGAAAACCACTGCCAGGTCACGCA TCAGCAAGACCAAAGGCCAGCCCCGC TGAAGGGAGCACCGTGAGCCCCAGGTGTACACACTGCCTCC GGAGAAGACAGTGG AAGCCGGGAAGAGATGACCAAGAACCCCCTGCAGAATGCT CAGGTGTCCCTGACCTGTCTCGTGAA CT (SEQ ID NO: 19)AGGCTTCTACCCCTCCGATATCGCCGT GGAATGUGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCC CATGCTGGACAGCGCGGCTCATTCTTCCTGTACAGCAAGCTCTACAGTGGACAA GTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAAGCC CTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGC (SEQ ID NO: 20)

In some embodiments, isolated nucleic acid molecules encode an antibodythat binds to ATβH and inhibits anticoagulant activity but has minimalbinding to ATα, wherein the antibody comprises a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, and 10.

In some embodiments, isolated nucleic acid molecules encode an antibodythat binds to ATβH and inhibits anticoagulant activity but has minimalbinding to ATα, wherein the antibody comprises a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7, and 9.

In other embodiments, isolated nucleic acid molecules encode an antibodythat binds to ATβ and inhibits anticoagulant activity of ATβ, whereinthe antibody comprises a heavy chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6,8, and 10 or a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7,and 9 and one or more amino acid modifications in the heavy chainvariable region or light chain variable region.

Further, also provided are vectors comprising the isolated nucleic acidmolecules encoding any of the monoclonal antibodies described above andhost cells comprising such vectors.

Methods of Preparing Antibodies to ATβH

The monoclonal antibody can be produced recombinantly by expressing anucleotide sequence encoding the variable regions of the monoclonalantibody according to one of the present embodiments in a host cell.With the aid of an expression vector, a nucleic acid containing thenucleotide sequence can be transfected and expressed in a host cellsuitable for the production. Accordingly, an exemplary method forproducing a monoclonal antibody that binds with human ATβH can comprise:(a) transfecting a nucleic acid molecule encoding a monoclonal antibodyinto a host cell; (b) culturing the host cell so to express themonoclonal antibody in the host cell, and (c) optionally isolating andpurifying the produced monoclonal antibody, wherein the nucleic acidmolecule comprises a nucleotide sequence encoding a monoclonal antibody.

In one example, to express the antibodies, or antibody fragmentsthereof; DNAs encoding partial or full-length light and heavy chainsobtained by standard molecular biology techniques are inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” refers to an antibody gene that is ligatedinto a vector such that transcriptional and translational controlsequences within the vector serve their intended function of regulatingthe transcription and translation of the antibody gene. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vectors or,alternatively, both genes are inserted into the same expression vector.The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the VH segmentis operatively linked to the CH segments) within the vector and the VLsegment is operatively linked to the CL segment within the vector.

Additionally, or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein). In addition to the antibody chainencoding genes, the recombinant expression vectors carry regulatorysequences that control the expression of the antibody chain genes in ahost cell. The term “regulatory sequence” includes promoters, enhancers,and other expression control elements (e.g., polyadenylation signals)that control the transcription or translation of the antibody chaingenes. Such regulatory sequences are described, for example, in Goeddel;Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Examples of regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,non-viral regulatory sequences can be used, such as the ubiquitinpromoter or β-globin promoter.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors can carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Examples of selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection”encompasses a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection, and the like. Although it is theoreticallypossible to express the antibodies in either prokaryotic or eukaryotichost cells, expression of antibodies in eukaryotic cells, includingmammalian host cells, is typical because such eukaryotic cells, and inparticular mammalian cells, are more likely than prokaryotic cells toassemble and secrete a properly folded and immunologically activeantibody. Examples of mammalian host cells for expressing therecombinant antibodies include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells, HKB11 cells and SP2 cells.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies arc produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods, such as ultrafiltration, size exclusionchromatography, ion exchange chromatography and centrifugation.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chain CDRs.For this reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al.,1986, Nature 321:522-525; and Queen, C. etal., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences. These germline sequences will differfrom mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. It is not necessary to obtain the entire DNAsequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see WO 99/45962).

Partial heavy and light chain sequence spanning the CDR regions istypically sufficient for this purpose. The partial sequence is used todetermine which germline variable and joining gene segments contributedto the recombined antibody variable genes. The germline sequence is thenused to fill in missing portions of the variable regions. Heavy andlight chain leader sequences are cleaved during protein maturation anddo not contribute to the properties of the final antibody. For thisreason, the corresponding germline leader sequence is used forexpression constructs. To add missing sequences, cloned cDNA sequencescan be combined with synthetic oligonucleotides by ligation or PCRamplification. Alternatively, the entire variable region can besynthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons. The nucleotide sequences of heavy and light chain transcriptsare used to design an overlapping set of synthetic oligonucleotides tocreate synthetic V sequences with identical amino acid coding capacitiesas the natural sequences. The synthetic heavy and light chain sequencescan differ from the natural sequences. For example, strings of repeatednucleotide bases are interrupted to facilitate oligonucleotide synthesisand PCR amplification; and optimal translation initiation sites areincorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem.266:19867-19870); and restriction sites are engineered upstream ordownstream of the translation initiation sites. For both the heavy andlight chain variable regions, the optimized coding, and correspondingnon-coding, strand sequences are broken down into 30-50 nucleotidesections at approximately the midpoint of the corresponding non-codingoligonucleotide. For each chain, the oligonucleotides can be assembledinto overlapping double stranded sets that span segments of 150-400nucleotides. The pools are then used as templates to produce PCRamplification products of 150-400 nucleotides.

Typically, a single variable region oligonucleotide set will he brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It can alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs. Thereconstructed heavy and light chain variable regions are then combinedwith cloned promoter, translation initiation, constant region, 3′untranslated, polyadenylation, and transcription termination sequencesto form expression vector constructs. The heavy and light chainexpression constructs can be combined into a single vector,co-transfected, serially transfected, or separately transfected intohost cells which are then fused to form a host cell expressing bothchains. In another aspect, the structural features of a human anti-ATβHantibody are used to create structurally related human anti-ATβHantibodies that retain the function of binding to ATβ. For example, oneor more CDRs of the specifically identified heavy and light chainregions of the monoclonal antibodies can be combined recombinantly withknown human framework regions and CDRs to create additional,recombinantly-engineered, human anti-ATβH antibodies.

Pro-Coagulant Efficacy of Anti-ATβH mAbs

Pro-coagulant efficacy of anti-ATβH mAbs was investigated using variousassays.

Table 4 shows pro-coagulant efficacy of Anti-ATβH mAbs TPP2009 andTPP2803 in plasma from various animal species in the FXa-activatedclotting assay.

TABLE 4 Pro-coagulant efficacy of TPP2009 and TPP2803 in plasma fromvarious animal species. HEM A HEM A Normal Plasma Plasma Plasma (EC₅₀nM) (EC₅₀ in nM) (EC₅₀ in nM) Species 2009 2803 2009 2803 2009FXa-Activated Clotting Assay dPT Human 10.5 2.4 4.7 2.7  9.0 Mouse NDRND ND ND NDR Rat NDR ND ND ND ND Rabbit 25.7 ND 10.5 ND NDR Beagle NDRND ND ND NDR Cyno 21.5 ND 4.3 ND 13.9 NDR: no dose response, ND: notdetermined, dPT: diluted prothrombin time, HEM-A: Hemophilia A plasma.

Anti-ATβH mAbs TPP2009 and TPP2803 both exhibited pro-coagulant efficacyin human normal plasma and hemophilia A plasma in the FXa-activatedclotting assay. Specifically, TPP2009 exhibited pro-coagulant efficacyin human normal plasma and hemophilia A plasma in the FXa-activatedclotting assay with EC₅₀'s of 10.5 nM and 4.7 nM, respectively. TPP2803exhibited pro-coagulant efficacy in human normal plasma and hemophilia Aplasma in the FXa-activated clotting assay with EC₅₀'s of 2.4 and 2.7nM, respectively.

In addition, Anti-ATβH mAb TPP2803 exhibited dose-dependent shorteningof the clotting time in both normal human plasma and hemophilia patientplasma using the FXa activated clotting assay, as shown in FIG. 12. CT:clotting time, HEM-A: Hemophilia A plasma.

The heparinized rabbit bleeding model outlined in FIG. 13 was employedto demonstrate in vivo pro-coagulant efficacy of Anti-ATβH mAb TPP2803.The effect of a control and TPP2803 on bleeding time before and afterLMWH and compound administration in a heparinized rabbit bleeding modelare shown in FIG. 14. Bleeding time after administration of LMWH andTest Article (either 3 mg/kg or 30 mg/kg TPP2803) was significantlyreduced compared to bleeding time after LMWH in PBS.Delta bleeding timeafter administration of either Test Article (either 3 mg/kg or 30 mg/kgTPP2803), or positive control protamine sulfate, was significantlydifferent from PBS (p≤0.05; * significance by T-test) as shown in FIG.15. The effect of a control and TPP2803 on blood loss before and afterLMWH and antibody administration is shown in FIG. 16. TPP2803 (3 mg/kg)and positive control protamine sulfate both exhibited a significantchange in blood loss (*Significance by the T-test) after administrationof LMWH.

Pharmaceutical Compositions

Also provided are pharmaceutical compositions comprising therapeuticallyeffective amounts of anti-ATβH monoclonal antibody and apharmaceutically acceptable carrier. “Pharmaceutically acceptablecarrier” as used herein refers to a substance that can be added to theactive ingredient to help formulate or stabilize the preparation andcauses no significant adverse toxicological effects to the patient.Examples of such carriers are well known to those skilled in the art andinclude water, sugars such as maltose or sucrose, albumin, salts such assodium chloride, etc. Other carriers are described for example inRemington's Pharmaceutical Sciences by E. W. Martin. Such compositionswill contain a therapeutically effective amount of at least onemonoclonal antibody.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art. Thecomposition is in some embodiments formulated for parenteral injection.The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. In some cases, the composition of the carrierincludes isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, some methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Pharmaceutical Uses

The monoclonal antibody can be used for therapeutic purposes fortreating genetic and acquired deficiencies or defects in coagulation.For example, the monoclonal antibodies in the embodiments describedabove can be used to block the interaction of ATβH with its substrate,which can include Factor Xa or Factor IIa. The monoclonal antibodieshave therapeutic use in the treatment of disorders of hemostasis such asthrombocytopenia, platelet disorders and bleeding disorders (e.g.,hemophilia A, hemophilia B and hemophilia C). Such disorders can betreated by administering a therapeutically effective amount of theanti-ATβH monoclonal antibody to a patient in need thereof. Themonoclonal antibodies also have therapeutic use in the treatment ofuncontrolled bleeds in indications such as trauma and hemorrhagicstroke. Thus, also provided is a method for shortening the bleeding timecomprising administering a therapeutically effective amount of ananti-ATβH monoclonal antibody to a patient in need thereof.

In another embodiment, the anti-ATβH antibody can be useful as anantidote for AT treated patients, including for example wherein AT isused for the treatment of sepsis or bleeding disorder.

The antibodies can be used as monotherapy or in combination with othertherapies to address a hemostatic disorder. For example,co-administration of one or more antibodies with a clotting factor suchas Factor VIIa, Factor VIII or Factor IX is believed useful for treatinghemophilia. In at least some embodiments, a method for treating geneticand acquired deficiencies or defects in coagulation comprisesadministering: (a) a first amount of a monoclonal antibody that binds tohuman tissue factor pathway inhibitor; and (b) a second amount of FactorVIII or Factor IX, wherein said first and second amounts together areeffective for treating said deficiencies or defects. In at least someembodiments, a method for treating genetic and acquired deficiencies ordefects in coagulation comprises administering: (a) a first amount of amonoclonal antibody that binds to human tissue factor pathway inhibitor;and (b) a second amount of factor VIII or Factor IX, wherein said firstand second amounts together are effective for treating said deficienciesor defects, and further wherein Factor VII is not co-administered. Alsoprovided is a pharmaceutical composition comprising a therapeuticallyeffective amount of the combination of a monoclonal antibody and FactorVIII or Factor IX, wherein the composition does not contain Factor VII.“Factor VII” includes Factor VII and Factor VIIa. These combinationtherapies are likely to reduce the necessary infusion frequency of theclotting factor. By co-administration or combination therapy is meantadministration of the two therapeutic drugs each formulated separatelyor formulated together in one composition, and, when formulatedseparately, administered either at approximately the same time or atdifferent times, but over the same therapeutic period.

In some embodiments, one or more antibodies described herein can be usedin combination to address a hemostatic disorder. For example,co-administration of two or more of the antibodies described herein isbelieved useful for treating hemophilia or other hemostatic disorder.

The pharmaceutical compositions can be parenterally administered tosubjects suffering from hemophilia A or B at a dosage and frequency thatcan vary with the severity of the bleeding episode or, in the case ofprophylactic therapy, can vary with the severity of the patient'sclotting deficiency.

The compositions can be administered to patients in need as a bolus orby continuous infusion. For example, a bolus administration of anantibody as a Fab fragment can be in an amount from about 0.0025 toabout 100 mg/kg body weight, about 0.025 to about 0.25 nag/kg, about0.010 to about 0.10 mg/kg or about 0.10 to about 0.50 mg/kg. Forcontinuous infusion, an inventive antibody present as an Fab fragmentcan be administered at about 0.001 to about 100 mg/kg bodyweight/minute, about 0.0125 to about 1.25 mg/kg/min, about 0.010 toabout 0.75 mg/kg/min, about 0.010 to about 1.0 mg/kg/min, or about 0.10to about 0.50 mg/kg/min for a period of about 1-24 hours, about 1-12hours, about 2-12 hours, about 6-12 hours, about 2-8 hours, or about 1-2hours. For administration of an inventive antibody present as afull-length antibody (with full constant regions), dosage amounts can beabout 1-10 mg/kg body weight, about 2-8 mg/kg, or about 5-6 mg/kg. Suchfull-length antibodies would typically be administered by infusionextending for a period of thirty minutes to three hours. The frequencyof the administration would depend upon the severity of the condition.Frequency could range from three times per week to once every two weeksto six months.

Additionally, the compositions can be administered to patients viasubcutaneous injection. For example, a dose of about 10 to about 100 mganti-ATβH antibody can be administered to patients via subcutaneousinjection weekly, biweekly or monthly. As used herein, “therapeuticallyeffective amount” means an amount of an anti-ATβH monoclonal antibody orof a combination of such antibody and Factor VIII or Factor IX that isneeded to effectively increase the clotting time in vivo or otherwisecause a measurable benefit in vivo to a patient in need thereof. Theprecise amount will depend upon numerous factors, including thecomponents and physical characteristics of the therapeutic composition,intended patient population, individual patient considerations, and thelike, and can readily be determined by one skilled in the art.

Aspects of the present disclosure may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLE Human and Rabbit ATα and ATβ Purification

ATα and ATβ were purified from human and rabbit plasma by affinitychromatography on heparin-sepharose according to methods previouslydescribed (Carlson and Atencio 1982; Peterson and Blackburn 1985) atEnzyme Research laboratory' (South Bend, Ind.). Briefly, the supernatantfrom a dextran sulphate/calcium chloride precipitation was applied to aheparin-sepharose affinity column (Pharmacia). ATα and ATβ wereseparated with a NaCl gradient: ATα and ATβ were eluted at 0.8 M and 1.3M NaCl, respectively. Anion-exchange chromatography (HiTrap-Q,Pharmacia) was employed for further purification of ATβ. Purity andglycan profile of ATα and ATβ were evaluated by protein SDS-PAGE andLC-MS.

EXAMPLE 2 Determination of the Number and Position of Glycans on ATα andATβ by Mass-Spectrometry Analysis

Due to the distinct number of glycans, ATα and ATβ were differentiatedbased on their mass by Agilent 6520 LC-MS system which is equipped withduo-ESI (or nano ChipCube) source, MassHunter acquisition software andqualitative analysis software including Bioconfirm. Glycosylation siteswere determined by a bottom-up method in which proteins are digested bytrypsin and Arg-c followed by target MSMS to identify the glycosylatedand mono-glycosylated peptide sequences. Data was collected in twoexperiments: Fragmentor voltage 175v and 430v.

EXAMPLE 3 AT Antigen Biotinylation

Human and rabbit ATα and ATβ were labeled with biotins on the surfacelysine residues by NHS-biotin. For lysine biotinylation, proteins werefirst desalted into PBS/Ca⁺⁺ buffer (Life Technologies Corporation,Carlsbad, Calif.) to remove any amines that might be inhibitory to thebiotinylation reaction. Concentrations of desalted proteins weredetermined by OD280 on the NanoDrop. Protein were then incubated for 1hour at room temperature (RT) with Sulfo-NHS-Biotin (Pierce ThermoScientific, Rockford, Ill.) at the 1:5 and/or 1:3 molar ratio ofAT:NHS-biotin (i.e. biotin in excess). Free biotin was removed byovernight dialysis into PBS/Ca⁺⁺ buffer. The amount of biotin in thebiotinylated proteins was quantified using Biotin Quantitation Kit(Pierce Thermo Scientific, Rockford, Ill.). Biotinylated ATα and ATβwere analyzed by SDS-PAGE, and biotinylation was confirmed by Westernblot analysis using streptavidin-HRP (Pierce Thermo Scientific,Rockford, Ill.) as probe. The functional activities of biotinylated ATwere evaluated by FXa inhibition assay. By comparison of thebiotinylated ATα and ATβ with unbiotinylated ATα and ATβ, only slightreductions in AT inhibition activity were observed after biotinylation,indicating the biotinylated ATβ and ATα prepared in this way would berepresentative and could be used in panning for ATβH binders asselective anti-coagulant blockers.

EXAMPLE 4 Human Monoclonal Antibody Discovery by Phage Display andPanning

A four-arm panning strategy was designed to discover Fabs specificallyagainst ATβH from a human Fab library (Dyax Fab310). The library wasfirst depleted with biotinylated heparin/Fondaparinux-bound ATα andbiotinylated ATα and then was panned against heparin/Fondaparinux-boundATβ and biotinylated ATβ on streptavidin beads, respectively. For eachround of panning, the heparin-bound ATα (ATαH) was included in thebinding buffer as a competitor. To keep hATβ in active conformation(heparin bound form), heparin was added to the wash buffer in all threerounds of panning. After panning, pooled clones were screened for hATβand hATβH specific binding and counter-screened for hATα by ELISA. Theseclones were also examined for differential binding to rabbit ATβ overrabbit ATα. Clones showing differential binding to both hATβH and rATβHover hATα and rATα were further subject to FXa—deinhibition assay withhATβ spike-in. Positive hits (Fabs) were reformatted into IgG1,expressed in HEK293 cells and purified by protein-A column. Thesepurified IgG1s were extensively tested in AT-depleted human plasma andhemA patient plasma for TGA assay (Thrombin Generation Assay) and dPT(diluted Prothrombin Time) assay to measure the clotting time.

EXAMPLE 5 ELISA (Enzyme-Linked Immunosorbent Assay)

2 ug/ml biotinlyated AT antigens in PBS were coated on StreptavidinMicroplates (Greiner, 781997) with or without heparin (50 ug/ml,heparin-Natrium-5000, Apotheke, Fa. Ratiopharm). After overnight antigencoating at 4° C., plates were washed with PBST+/−heparin and blockedwith 5% milk in PBST+/−heparin at 37° C. for one hour. After removal ofblocking buffer, 20 ug/ml Fab or 4 ug/ml IgG in blocking buffer (5% milkin PBST+/−heparin) was then added to the plates and plates wereincubated at room temperature for 1 hour. Plates were then washed threetimes. Anti-human IgG POD (Sigma, A0170) in blocking buffer was added toplates and plates were incubated at room temperature for 30 minutes.Amplex red (In vitrogen, Cat #A22170) was used for detection at 1:1000together with H₂O₂. After 30 min incubation, plates were read at Ex535,Em 590 in a fluorescent plate reader.

EXAMPLE 6 FXa De-Inhibition Assay—AT With Heparin

Heparin was incubated with ATβ or ATα to form stable ATβH complexes.Antibody was then added to the ATβH or ATαH complexes. In the meantime,10 μl of 200 ng/ml FXa (HTI) and 20 μl of 50 μg/ml Fluophen FXafluorogenic substrate (Hyphen Biomed) were mixed in a separate plate.The antibody-ATβH mixture was added to the FXa/substrate solutionquickly and fluorescent kinetic measurement was started immediately atEx360 nm and Em465 nm. All necessary dilutions is made in 100 mM NaCl,20 mM Tris, 2.5 mM CaCl₂, 0.1% BSA, 0.1% PEG8000.

EXAMPLE 7 Thrombin Generation Assay (TGA) in FVIII Deficiency HumanPlasma

A 1:2 serial dilution of ATβH antibody was made in HemA human plasmastarting from 1 uM of final concentration to 0.015 uM. Heparin was addedin each antibody solution at a final concentration of 50 nM. An 80 ul ofthe antibody-heparin-plasma mixture was then added to each wellcontaining 20 ul of reconstitute PPP reagent or calibrator in a 96 wellTGA plate. The plate was placed in the TGA instrument and the machineautomatically dispensed 20 ul of FluCa (Fluo substrate+CaCl₂) into eachwell. The reaction was allowed to run 60 min. Plasma alone was used asthe negative control.

EXAMPLE 8 Thrombin Generation Assay (TGA) in AT-Depleted Human PlasmaWith Spiked-In ATα and ATβ Respectively

Antibodies were added to human AT-deficient plasma spiked with 15 nm ofATα or ATβ. Heparin was then pipetted into each reaction at a finalconcentration of 50 nM. 80 ul of plasma samples containing ATH-specificantibody, heparin and ATα or ATβ were added into wells of a 96 well TGAplate with 20 ul of PPP reagent or calibrator. Plates were placed in theTGA instrument, and then 20 ul of FluCa (Fluo substrate+CaCl₂) wasdispensed into each well. Reactions were allowed to continue for 60 min.

EXAMPLE 9 Diluted Prothrombin Time Assay (dPT) in Human hemA Plasma andAT Deficient Plasma

A serial dilution of anti-ATβH hmAbs was made in hemA plasma starting at250 nM with 0.1 U/mL of heparin. The mixture of antibody, plasma andheparin was incubated at room temperature for 20-30 min. Then 50 uL ofthis mixture was added to a 50 uL of diluted Innovin (1/2000) (DadeBehring), incubated for 4 min at 37° C., followed by adding 50 uL of 25mM CaCl2 (HemSil). dPT test program was set on ACL Top coagulometer withacquisition time of 360 seconds. For dTP in AT deficient plasma, AT-DPwas spiked in with either ATα or ATβ at a final concentration of 0.2 uMwith 0.1 U/ml of heparin. Anti-ATβH mAbs was added to AT-DP/heparin/ATαor AT-DP/heparin/ATβ mixtures at a final concentration of 0.25 uM andincubated at room temperature for 20-30 min. For each reaction, a 50 uLof plasma/antibody/heparin mixture was added to 50 uL of diluted Innovin(1/4000), incubate 4 min at 37° C., followed by adding 50 uL of 25 mMCaCl2 (HemSil) as above.

EXAMPLE 10 Antibody Purification

Pre-washed protein A agarose beads were incubated with antibody inbinding buffer (volume ratio: 1:1) with rotation overnight at 4° C.Beads were then packed into a column and washed with 1× PBS untilO.D.₂₈₀<0.05. Residual solution was drained. Antibodies were eluted withelution buffer and collected into tubes containing neutralizing buffer.Eluted fractions were dialyzed against 1× PBS overnight at 4° C. with atleast twice buffer changes. IgG concentration was measured at 280 nm bynanodrop. The antibody purity was examined by either ELISA, SDS-PAGE orSSC.

EXAMPLE 11 Antibody Binding Affinity Study by Biacore

Antibody affinity measurement was performed on a Biacore T100 or T200processing unit. Anti-human Fc antibody or streptavidin was immobilizedon a CM5 chip. hATβH or biotinylated hmAb antibodies were injected andcaptured on the chip. ATβ or ATα at different concentration with/withoutheparin were injected. Only AT and ATH bound to the antibodies generatebinding constants. The binding results were reported as EquilibriumDissociation Constants (KD) in nanoMoles. When AT/heparin complex wasanalyzed, heparin at 1 uM is included in the running buffer.

EXAMPLE 12 Heparinized Rabbit Bleeding Model

Experimental design of the heparinized rabbit bleeding model is outlinedin FIG. 13. Following preparation of rabbit jugular veins (right vein:venous stasis; left vein: cannulation), low molecular weight heparin(LMWH) is administered to the rabbit (1800 U/kg) IV in PBS vehicle attime 0. After 10 minutes, the test article is administered. Experimentalgroups include Vehicle, PBS; Positive control, Protamine sulfate, (28mg/kg IV); Negative control, M14 IgG2; treatment: 30 mg/kg; TPP2803, 3mg/kg; TPP2803, 30 mg/kg. Five minutes after administration of testarticle, an ear puncture (3-5 mm) is performed and thrombus formation insitu (stasis) is monitored over a 30 min period. Blood from the incisionis removed every 15 seconds with a filter paper until the bleedingstops.

The foregoing disclosure and examples are not intended to narrow thescope of the claims in any way. It should be understood that variousmodifications and changes can be made, and equivalents can besubstituted, to the foregoing embodiments and teachings withoutdeparting from the true spirit and scope of the claims appended hereto.The specification and examples are, accordingly, to be regarded in anillustrative sense rather than in a restrictive sense. Furthermore, thedisclosure of all articles, books, patent applications, patents, andother material referred to herein are incorporated herein by referencein their entireties.

1.-21. (canceled)
 22. A method for treating coagulopathy comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a monoclonal antibody capable of binding theantithrombin (β) heparin complex (ATβH), wherein: the heavy chain ofsaid antibody comprises: a CDR1 sequence of amino acids 31 to 35 (AYRMG)of SEQ ID NO: 2, a CDR2 sequence of amino acids 50 to 66(RIYSSGGRTRYADSVKG) of SEQ ID NO: 2, and a CDR3 sequence of amino acids97 to 114 (AREKASDLSGSFSEALDY) of SEQ ID NO: 2; and the light chain ofsaid antibody comprises: a CDR1 sequence of amino acids 24 to 34(QGDSLRSYYAS) of SEQ ID NO: 1, a CDR2 sequence of amino acids 50 to 56(GKNNRPS) of SEQ ID NO: 1; and a CDR3 sequence of amino acids 89 to 99(NSRDSSGNHLV) of SEQ ID NO:
 1. 23. The method of claim 22, wherein thecoagulopathy is hemophilia A, hemophilia B, or hemophilia C.
 24. Themethod of claim 22, wherein the coagulopathy is trauma-inducedcoagulopathy or severe bleeding.
 25. The method of claim 22, furthercomprising administering a clotting factor.
 26. The method of claim 25,wherein the clotting factor is selected from the group consisting ofFactor VIIa, Factor VIII, and Factor IX. 27.-30. (canceled)
 31. Themethod of claim 22, wherein the monoclonal antibody is an IgG1 antibody,an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgM antibody,an IgA1 antibody, an IgA2 antibody, a secretory IgA antibody, an IgDantibody, an IgE antibody, or an antibody fragment.
 32. A method fortreating coagulopathy comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising a monoclonalantibody capable of binding the antithrombin (β) heparin complex (ATβH),wherein: the heavy chain of said antibody comprises: a CDR1 sequence ofamino acids 31 to 35 (KYKMD) of SEQ ID NO: 4, a CDR2 sequence of aminoacids 50 to 66 (RIGPSGGKTM YADSVKG) of SEQ ID NO: 4, and a CDR3 sequenceof amino acids 97 to 114 (AREKASDLSG TYSEALDY) of SEQ ID NO: 4; and thelight chain of said antibody comprises: a CDR1 sequence of amino acids26 to 37 (RASQSVSSSYLA) of SEQ ID NO: 3, a CDR2 sequence of amino acids53 to 59 (GASSRAT) of SEQ ID NO: 3, and a CDR3 sequence of amino acids92 to 99 (QQYGSSRT) of SEQ ID NO:
 3. 33. The method of claim 32, whereinthe coagulopathy is hemophilia A, hemophilia B, or hemophilia C.
 34. Themethod of claim 32, wherein the coagulopathy is trauma-inducedcoagulopathy or severe bleeding.
 35. The method of claim 32, furthercomprising administering a clotting factor.
 36. The method of claim 35,wherein the clotting factor is selected from the group consisting ofFactor VIIa, Factor VIII, and Factor IX.
 37. The method of claim 32,wherein the monoclonal antibody is an IgG1 antibody, an IgG2 antibody,an IgG3 antibody, an IgG4 antibody, an IgM antibody, an IgA1 antibody,an IgA2 antibody, a secretory IgA antibody, an IgD antibody, an IgEantibody, or an antibody fragment.
 38. A method for treatingcoagulopathy comprising administering a therapeutically effective amountof a pharmaceutical composition comprising a monoclonal antibody capableof binding the antithrombin (β) heparin complex (ATβH), wherein: theheavy chain of said antibody comprises: a CDR1 sequence of amino acids31 to 35 (KYRMD) of SEQ ID NO: 6, a CDR2 sequence of amino acids 50 to66 (RIGPSGGKTT YADSVKG) of SEQ ID NO: 6, and a CDR3 sequence of aminoacids 97 to 114 (AREKTSDLSG SYSEALDY) of SEQ ID NO: 6; and the lightchain of said antibody comprises: a CDR1 sequence of amino acids 26 to36 (RASQNINRNLA) of SEQ ID NO: 5, a CDR2 sequence of amino acids 52 to58 (TASTRAP) of SEQ ID NO: 5, and a CDR3 sequence of amino acids 91 to99 (QQYASPPRT) of SEQ ID NO:
 6. 39. The method of claim 38, wherein thecoagulopathy is hemophilia A, hemophilia B, or hemophilia C.
 40. Themethod of claim 38, wherein the coagulopathy is trauma-inducedcoagulopathy or severe bleeding.
 41. The method of claim 38, furthercomprising administering a clotting factor.
 42. The method of claim 41,wherein the clotting factor is selected from the group consisting ofFactor VIIa, Factor VIII, and Factor IX.
 43. The method of claim 41,wherein the monoclonal antibody is an IgG1 antibody, an IgG2 antibody,an IgG3 antibody, an IgG4 antibody, an IgM antibody, an IgA1 antibody,an IgA2 antibody, a secretory IgA antibody, an IgD antibody, an IgEantibody, or an antibody fragment.